Biological membranes are permeable to exogenous opioid drugs--both plant-derived molecules such as morphine, and synthetic molecules of which hundreds exist. Now a genetically encoded fluorescent biosensor technique allows us to measure opioids within neutral organelles such as the endoplasmic reticulum (ER). We term these molecules the intensity-based opioid-sensitive fluorescent reporter, iOpioidSnFR, family (Figure 1). Ongoing experiments, before the project begins, will extend the iOpioidSnFR family to the major classes of -opioid agonists. Aim 1 Further extends the iOpioidSnFRs for measurements within acidic organelles such as endosomes and synaptic vesicles. Aim 1a utilizes the present circularly permutated green fluorescent protein (cpGFP) moiety. Aim 1b develops novel circularly permuted HaloTags, which are pH-insensitive. Aim 1c, Extends the existing measurements to measure the entry of opioids into organelles, and their exit from organelles. Quantification involves both dynamics and steady- state measurements. Aim 2 tests the hypothesis that some effects of opioid drugs result after synaptic vesicles accumulate opioids via acid trapping. The synaptic vesicles would then release the opioids upon presynaptic stimulation. This mechanism would extend the patho-pharmacology of exogenous opioids to their release from many types of presynaptic neurons?even those neurons that do not release endogenous opioid peptides. Aim 2a evolves iOpioidSnFR sensitivity further, to the required nanomolar levels. Aim 2b Identifies the most sensitive method for testing presynaptic release. Aim 3 tests the hypothesis that brain regions expressing -opioid receptors vary in the extent and timing of organellar opioids. Aim 3a generates adeno-associated viral vectors that encode ?floxed? iOpioidSnFRs. These will be expressed under the control of vesicular GABA transporter (vGAT) cre recombinase in suitable mouse lines. Aim 3b measures in brain slices from ventral tegmentum area (VTA) / substantia nigro pars reticulata (SnR), periaqueductal gray (PAG), and ventral pallidum (VP).The results will aid in the ongoing efforts to understand the cellular and molecular basis of tolerance to -opioid ligands.